Hydrogenase are used by many micro-organisms, including algae, to metabolize H2. During the metabolic energy-yielding process, H2 is either oxidized or evolved as the product. Hydrogenase is of interest not only because it is a natural organometallic catalyst but also because it has the potential application to biotechnological hydrogen production. Hydrogenase may also be used to improve the efficiency of nitrogen fixation by recycling the H2 produced during the fixation process, which in turn may increase agricultural productivity. Using EPR and Mossbauer techniques, we had successfully characterized the aspurified forms of hydrogenases isolated from C. pasteurianum, D. vulgaris, D. gigas, and D. desulfuricans (27774), and we had firmly established that hydrogenase can be grouped into two categories: (1) the [NiFe] hydrogenase which contains both Ni and Fe-S centers, and (2) the [Fe] hydrogenase which contains only Fe-S clusters. Combining EPR, Mossbauer, and redox-titration techniques, we were able to follow the changes ocurred in the metal centers in D. gigas hydrogenase during the redox cycle. Based on these studies, we have proposed a working hypothesis for the [NiFe] hydrogenase. We now propose to apply the same method to study the [Fe] hydrogenases isolated from C. pasteurianum and D. vilgaris. The studies include (1) examination of the time course of the H2 reduction of the enzymes, (2) determination of the mid-point redox potential of each metal center, and (3) investigation and characterization of the metal centers during the redox process. These studies should provide information concerning the changes on the physical properties of the Fe-S clusters in these [Fe] hydrogenases during their catalytic cycles. Such information is pertinent to the understanding of the mechanism of the [Fe] hydrogenase. We also propose series of systematic ligand-binding studies for both the [NiFe] and the [Fe] hydrogenases. CO and CN are chosen for these studies. EPR and Mossbauer spectroscopy will be used in conjunction with biochemical techniques. We also propose to extend our methods to include ENDOR and NMR. We expect to obtain information concerning the physiological functions of the Ni and the Fe-S clusters, and thus enhance our understanding of the hydrogenase mechanism.